Preparation of a slowly absorbed antidiabetic hormone



Patented May 2, 1939 Iv 's PREPARATION OF A SLOWLY ABSORBED I ANTIDIABETIC HORMONE 1 r Lazar Rosenthal and Jonas Kamlet, Brooklyn, N. Y.

Serial No. 111,617

No Drawing. Application November 19, 1936,

9 Claims.

The purpose .of this invention is to make possible the preparation of a slowly-absorbed antidiabetic hormone and, more exactly, to make possible the preparation of a slowly-absorbed antidiabetic hormone of such stability and purity as will render it useful in the treatment of diabetes mellitus in humans; The name universally accepted and commonly applied to the anti-diabetic hormone isinsulin and .it is understood, there- 1 fore, that the term anti-diabetic hormone and insulin, as used in this specification and claims, are identical, synonymous and freely interchangeable and that all claims made for the former are in every sense applicable to the latter, and vice versa.

The disadvantages and difficulties that are en'- countered in the use of ordinary insulin as it has been available heretofore, lie in the tendency of the human body to utilize with great rapidity any excessive dose of insulin injected, even subcutaneously or intradermally. The maximum reduction of blood glucose is reached in two to four hours after which the previous high blood sugar level is rapidly reattained. Every overdosage can beminimized or completely eliminated. A slowly-absorbed insulin most nearly imitates nature's own mechanism in non-diabetic mammals by slowly liberating and rendering substantially constant and uniform amounts of insulin available .for absorption by the blood stream. The advantages of slowly-absorbed insulin in comparison with the insulin preparations heretofore available would be:

* (1) The elimination of frequent injections. The total daily dosage of insulin could be given in one injection.

(2) The elimination of the constant menace of hypoglycemic tetany after an accidental overdosage.

(3) A more uniform blood sugar level throughout the day and night. (4) A more prolonged lowering of the blood sugar 55 level by the new preparation.

produce 'a so-called insulin shock or hypo--- In order to effect such a retarded absorption, suspensions or emulsions of insulin in oil have been used by Leyton (Lancet, Vol. I, pg. 756, 1929).

Such injections are usually painful and, constant- (1) The difiiculty of obtaining a substantially pure preparation of protamine. 5

(2) The necessity of introducing daily into the 1 body for considerable periods of time large amounts of a foreign protein which may have undesirable effects.

(3) The necessity of preparing ex tempore the mixture of protamine and insulin due to the instability of the preparation thus obtained. Besides the inconvenience to the patient, this is conducive to errors and occurrences of contamination and infection.

Our invention is based on an entirely new and previously undescribed property of insulin. We find that by adding an aqueous solution of a compound of thetype MA1(SO4)2.12H2O, whereas M represents sodium, potassium, lithium or ammonium, to an aqueous solution or suspension of insulin at a pH between 1 and 7, there is obtained a copious precipitate of a compound of insulin and alum which, when suspended in an aqueous medium and injected subcutaneously or intradermally will be gradually and slowlyabsorbed by the blood stream. In contradistinction to the usual insulin heretofore available which is absorbed rapidly'from the subcutaneous 4 tissue and produces the maximum depression of the blood sugar level in one to three hours, this new alum-precipitated insulin will cause' the maximum decrease to occur in seven to ten hours and complete recovery to the fasting blood sugar level to be observed in fifteen to twenty-four.

Alum-precipitated insulin is a white, amorphous powder, insoluble in alcohol, ether and petroleum ether and slightly soluble in neutral (pH 7) distilled water and physiological saline.

It is stable in aqueous fluids of pH 2.5 to 7.0, but dissolves completely in solutions of acidity greater than pH 2.5. When dissolved in dilute acids (pH 2.5 orbelow) alum-precipitated insulin reacts in every way like ordinary insulin hydrochloride. Therefore, in order to assay physiologically a preparation of our new slowly absorbed antidiabetic hormone, it is only necessary to adjust it to pH '1 to 2. The number of units available in the resultant clear solution of insulin can be determined in the usual manner in accordance with the well-established international standards.

Chemical analysis of the alum-precipitated insulin as prepared by us shows a concentration of 0.10 gamma to 0.12 gamma (0.00010 to 0.00012 mgms.) of aluminum and 4.0 gamma to 7.0 gamma (0.004 to 0.007 mgms.) of non-protein nitrogen per unit. It is readily soluble in undilutedlserum containing 25% to 30% of protein but only slightly soluble in serum diluted 1:5 with physiological saline. In solutions of pH 3 to '7, it forms insoluble suspensions, while the optimum suspensions are obtained in solutions of pH 6. Above pH '7, there is a gradual loss in the potency of alum-precipitated insulin.

It is advisable, therefore, to dispense and use alum-precipitated insulin suspended in sterile aqueous media of pH 3 to pH '7, preferably pH 6, preserved in the, usual manner by the addition of weak preservatives Preparations of alum-precipitated insulin suspended in phenolized aqueous solutions of pH 6 and containing twenty, forty and eighty alum-precipitated insulin units per cc. have been kept'by us without any evidence of decrease in potency for six months in a refrigerator at 5-l0 C.

Our experiments have shown that our preparation is slowly absorbed from the subcutaneous tissue, is non-toxic on prolonged and repeated administration, does not form infiltrates or irri-' tate the tissue on the site of the injection, causes a gradual and prolonged lowering of the blood sugar level and relieves all the cardinal objective symptoms of diabetes mellitus in hiunans.

The following example is intended to define and illustrate this invention, but in no way limit it to the reagents, proportions or conditions described therein. Obvious modifications and improvements will occur to any person skilled in the art.

As the starting material for the preparation of alum-precipitated insulin we prefer to use ordinary insulin solution as it is at present available for use.

To 1000 cc. of insulin solution assaying 40 units per cc., there is added 80 cc. of a 50% by weight solution of sodium aluminum sulphate dodecahydrate (NaAllSOrlal2I-Iz0) in sterile distilled water. A heavy turbidity forms instantly and, upon mixing, a copious precipitation of alumprecipitated insulin'occurs. After standing for' two hours in a refrigerator at 5-10 C., the reaction mixture is centrifuged at'3500 R. P. M. for- 30 minutes. The clear supernatant fluid is now quantitatively decanted from the sediment of the major portion of the alu -precipitated insulin.

To this supernatant fluid is now added a furabsorbed anti-diabetic hormone which ther portion of cc. of a 50% by weight solution of sodium alum and, after mixing, it is allowed to stand in a refrigerator at 5-10 C. for fortyeight hours. A second crop of alum-precipitated insulin is thus obtained, separated from the supernatant fluid by centrifuging at 3500 R. P. M. for one hour and decanting. The clear supernatant fluid, without further addition of alum will yield a third crop of alum-precipitated insulin on being allowed to stand in the refrigerator at 5-10 C. for two weeks. In this manner, a quantitative recovery of the insulin originally present in solution may ultimately be obtained as alum-precipitated insulin.

The combined sediments of alum-precipitated insulin are now suspended in 1000 cc. of a solution containing 0.1' cc. of one-tenth normal hydrochloric acid and 0.2% of phenol per liter of steril distilled water. This suspension may be readily dispensed in vials and ampules for use by the diabetic patient and assays 40 units of alumprecipitated insulin per cc.

Having thus fully described our invention, what we claim as new and desire to protect by Letters Patent is:

1. A process for the preparation of slowlyabsorbed anti-diabetic hormone which comprises reacting an aqueous suspension or solution of insulin at a pH between 1 and 7, with an aqueous solution of an alum and suspending the resultant precipitate in an aqueous medium.

2. A- process for the preparation of slowlycomprises reacting an aqueous suspension or solution of insulin at a pH- between 1 and '7, with an aqueous solution of an alum,.and suspending the resultant precipitate in aqueous media of pH 3 to 7.

3. A process for the preparation of slowlyabsorbed anti-diabetic hormone which comprises reacting an aqueous suspension or solution of insulin at a pH between 1 and '7, with an aqueous solution of an alum, and suspending the resultant precipitate in aqueous media of pH 6.

4. As a new group of compounds, the double salts of insulin with alums;

5. As a new slowly-absorbed anti-diabetic hormone, the compounds of insulin with alums at 

